The central vacuolar system of eukaryotic cells is an interlocking membrane system composed of distinct organelles (including ER, Golgi apparatus, secretory vesicles, secretory granules, plasma membrane, endosomes, and lysosomes) and selective transport pathways. Proper functioning of this system is vital for regulating surface expression and secretion of proteins. Current work in this project is aimed at understanding three distinct aspects of the regulation and mediation of traffic within the organelle system. Work over the past year has seen a shift in our focus from the secretory pathway to the peripheral endocytic pathway. This work is divided into three subprojects: (1) The role of ARF family GTP-binding proteins and the regulation of membrane traffic into and out of the cell surface. We have identified that a distinct subfamily of ARF GTP-binding proteins appears to regulate the critical modeling events that determine the movement of membrane into the cell and back to the plasma membrane. (2) The ability of specific proteins to move into various compartments of the central vacuolar system depends upon the expression of simple targeting motifs within the primary sequences of those proteins. Two years ago, this laboratory had identified a new generic class of targeting signals based upon the presence of a double leucine. These sequences, termed dileucine motifs, are involved in the targeting of numerous membrane proteins to a variety of specific fates within the peripheral membrane system. We have been carefully analyzing the sequence, context, and structural properties of several dileucine motifs in order to begin to understand the nature of their targeting abilities and targeting specificities. (3) Over the past year, we have developed a new genetic model system in the simple yeast Saccharomyces cerevisiae in order to begin to understand the process of endocytosis and membrane protein turnover in this organism. This system uses the recent discovery in CBMB of a plasma membrane metal transporter whose internalization and degradation is controlled by copper. This phenomenon allows us to establish biochemical, morphological, and genetic approaches to identifying the gene products required for plasma membrane protein internalization and degradation.